The present invention relates to compounds of formula (I), which are useful for treating diseases or conditions caused by or exacerbated by P2X receptor activity, pharmaceutical compositions containing compounds of formula (I) and methods of treatment using compounds of formula (I).
P2X receptors function as homomultimeric cation-permeable ion channels and, in some cases, as heteromeric channels consisting of two different P2X receptor subtypes. At least one pair of P2X receptor subtypes, P2X2 and P2X3, functions as a heteromeric channel in rat nodose ganglion neurons where it exhibits distinct pharmacological and electrophysiological properties.
With respect to individual receptors, the rat P2X2 containing receptor is expressed in the spinal cord, and in the nodose and dorsal root ganglia, while rat P2X3 containing receptor expression is found primarily in a subset of neurons of the sensory ganglia. The distribution of both receptors is consistent with a role in pain transmission. The P2X2 and P2X3 subunits form functional channels when expressed alone, and can also form a functional heteromultimeric channel that has properties similar to currents seen in native sensory channels when co-expressed. Evidence from studies in rat nodose ganglia indicate that both P2X2/P2X3 heteromeric channels and P2X2 homomeric channels contribute to adenosine triphosphate-induced currents.
ATP, which activates P2X2, P2X3, and P2X2/P2X3 containing receptors, functions as an excitatory neurotransmitter in the spinal cord dorsal horn and in primary afferents from sensory ganglia. ATP-induced activation of P2X receptors on dorsal root ganglion nerve terminals in the spinal cord stimulates the release of glutamate, a key neurotransmitter involved in nociceptive signaling. Thus, ATP released from damaged cells can evoke pain by activating P2X2, P2X3, or P2X2/P2X3 containing receptors on nociceptive nerve endings of sensory nerves. This is consistent with the induction of pain by intradermally applied ATP in the human blister-base model; the identification of P2X3 containing receptors on nociceptive neurons in the tooth pulp; and with reports that P2X antagonists are analgesic in animal models. This evidence suggests that P2X2 and P2X3 function in nociception, and that modulators of these human P2X receptors are useful as analgesics.
It has been recently demonstrated that P2X3 receptor gene disruption results in a diminished sensitivity to noxious chemical stimuli and reduced pain. P2X3 containing receptor knock-out mice also exhibited a marked urinary bladder hyporeflexia upon cystometric evaluation, suggesting that P2X3 antagonists have utility for treating bladder overactivity. P2X3 knock-out mice had decreased voiding frequency, increased voiding volume, but normal bladder pressure. It has been proposed that ATP acts as a physiological regulator of sensory neurotransmission in visceral hollow organs such as bladder, and P2X3 containing receptors localized on the basal surface of the urothelium. The urology data on the P2X3 knock-out mice suggest that P2X3 plays a major role in modulating the volume threshold for activation of micturition and that P2X3 antagonists have therapeutic utility for urinary incontinence.
The nociceptive effects of exogenously administered ATP and P2X containing receptor agonists have also been demonstrated in laboratory animals. The peripheral nociceptive actions of P2X activation and stimulation of spinal P2X containing receptors also contribute to nociception as indicated by the ability of intrathecally (i.t.) administered P2 receptor agonists to increase sensitivity to acute and persistent noxious stimuli in rodents.
The utility of available purinergic ligands to evaluate the role of individual P2 receptor subtypes in mammalian physiology has been complicated by the susceptibility of P2 receptor agonists to undergo enzymatic degradation, and by the lack of P2 receptor subtype-selective agonists and antagonists.
Since subtype-selective ligands for the individual P2 receptors have yet to be identified, efforts to elucidate the specific P2X containing receptor subtypes involved in the transmission of nociceptive signals has been largely based on receptor localization and functional studies using immunohistochemical techniques. These studies have shown that both the homomeric P2X3 and heteromeric P2X2/3 containing receptor subtypes are selectively localized to the central and peripheral terminals of small diameter sensory neurons. Further, recent data has shown that P2X3 specific immunoreactivity is significantly increased in both the injured dorsal root ganglion and in the ipsalateral spinal dorsal horn following chronic constriction injury of the rat sciatic nerve.
The functional and immunohistochemical localization of P2X3 and/or P2X2/3 containing receptors on sensory nerves indicates that these P2X containing receptors have a primary role in mediating the nociceptive effects of exogenous ATP. Thus, compounds which block or inhibit activation of P2X3 containing receptors serve to block the pain stimulus. Antagonists of the P2X3 homomeric channel and/or the P2X2/P2X3 heteromeric channel could successfully block the transmission of pain.
The compounds of the present invention are novel P2X3 and P2X2/3 antagonists, have utility in treating pain as well as in treating bladder overactivity and urinary incontinence.
The present invention discloses fluorene and anthracene compounds, a method for controlling pain in mammals, and pharmaceutical compositions including those compounds. More particularly, the present invention is directed to compounds of formula (I) 
or a pharmaceutically acceptable salt, ester, amide, or prodrug thereof, wherein
A1 and A2 are independently selected from hydrogen or 
provided that one of A1 and A2 is hydrogen;
L1 and L2 are independently selected from N(R11) or S(O)2, provided that when L1 is N(R11) then L2 is S(O)2 or when L1 is S(O)2 then L2 is N(R11);
L3 is selected from S, S(O), S(O)2, C(O), CH(OR12), C(xe2x95x90NOR13), C(xe2x95x90NNR12R14), C(xe2x95x90CHC(O)OR12), CH2, or CH2CH2;
L4 is selected from a covalent bond, C(O), CH(OR12), C(xe2x95x90NOR13), or C(xe2x95x90NNR12R14);
L5 and L6 are independently selected from N(R15) or S(O)2, provided that when L5 is N(R15) then L6 is S(O)2 or when L5 is S(O)2 then L6 is N(R15);
R1, R2, R3, R4, R5, R6, R7, R8, R9, and R10 are independently selected from hydrogen, alkenyl, alkoxy, alkoxycarbonyl, alkyl, alkylcarbonyl, alkylcarbonyloxy, halogen, haloalkoxy, haloalkyl, hydroxy, or xe2x80x94NZ1Z2 wherein Z1 and Z2 are independently selected from hydrogen, alkyl, or alkylcarbonyl;
R11, R12, R14, and R15 are independently selected from hydrogen and alkyl; and
R13 is selected from hydrogen, alkyl, or carboxyalkyl.
All references contained herein are fully incorporated by reference.
In the principle embodiment, compounds of formula (I) are disclosed 
or a pharmaceutically acceptable salt, ester, amide, or prodrug thereof, wherein
A1 and A2 are independently selected from hydrogen or 
provided that one of A1 and A2 is hydrogen;
L1 and L2 are independently selected from N(R11) or S(O)2, provided that when L1 is N(R11) then L2 is S(O)2 or when L1 is S(O)2 then L2 is N(R11);
L3 is selected from S, S(O), S(O)2, C(O), CH(OR12), C(xe2x95x90NOR13), C(xe2x95x90NNR12R14), C(xe2x95x90CHC(O)OR12), CH2, or CH2CH2;
L4 is selected from a covalent bond, C(O), CH(OR12), C(xe2x95x90NOR13), or C(xe2x95x90NNR12R14);
L5 and L6 are independently selected from N(R15) or S(O)2, provided that when L5 is N(R15) then L6 is S(O)2 or when L5 is S(O)2 then L6 is N(R15);
R1, R2, R3, R4, R5, R6, R7, R8, R9, and R10 are independently selected from hydrogen, alkenyl, alkoxy, alkoxycarbonyl, alkyl, alkylcarbonyl, alkylcarbonyloxy, halogen, haloalkoxy, haloalkyl, hydroxy, or xe2x80x94NZ1Z2 wherein Z1 and Z2 are independently selected from hydrogen, alkyl, or alkylcarbonyl;
R11, R12, R14, and R15 are independently selected from hydrogen or alkyl; and
R13 is selected from hydrogen, alkyl, or carboxyalkyl.
In another embodiment of the present invention, compounds of formula (I) are disclosed wherein A2 is hydrogen; A1 is 
L1 and L5 are S(O)2; L2 is N(R11); L4 is a covalent bond; L6 is N(R15); and L3, R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R11, and R15 are as defined in formula (I).
In another embodiment of the present invention, compounds of formula (I) are disclosed wherein A2 is hydrogen; A1 is 
L1 and L5 are S(O)2; L2 is N(R11); L4 is a covalent bond; L6 is N(R15); R1, R3, R4, R5, R6, R8, R9, and R10 are hydrogen; R2 and R7 are independently selected from hydroxy, alkoxy, or xe2x80x94NZ1Z2; L3 is C(O); and Z1, Z2, R11, and R15 are as defined in formula (I).
In another embodiment of the present invention, compounds of formula (I) are disclosed wherein A2 is hydrogen; A1 is 
L1 and L5 are S(O)2; L2 is N(R11); L4 is a covalent bond; L6 is N(R15); R1, R3, R4, R5, R6, R8, R9, and R10 are hydrogen; R2 and R7 are independently selected from hydroxy, alkoxy, or xe2x80x94NZ1Z2; L3 is C(xe2x95x90NOR13); and Z1, Z2, R11, R13, and R15 are as defined in formula (I).
In another embodiment of the present invention, compounds of formula (I) are disclosed wherein A2 is hydrogen; A1 is 
L1 and L5 are S(O)2; L2 is N(R11); L4 is a covalent bond; L6 is N(R15); R1, R3, R4, R5, R6, R8, R9, and R10 are hydrogen; R2 and R7 are independently selected from hydroxy, alkoxy, or xe2x80x94NZ1Z2; L3 is C(xe2x95x90NNR12R14); and Z1, Z2, R11, R12, R14, and R15 are as defined in formula (I).
In another embodiment of the present invention, compounds of formula (I) are disclosed wherein A2 is hydrogen; A1 is 
L1 and L5 are S(O)2; L2 is N(R11); L4 is a covalent bond; L6 is N(R15); R1, R3, R4, R5, R6, R8, R9, and R10 are hydrogen; R2 and R7 are independently selected from hydroxy, alkoxy, or xe2x80x94NZ1Z2; L3 is CH(OR12); and Z1, Z2, R11, R12, and R15 are as defined in formula (I).
In another embodiment of the present invention, compounds of formula (I) are disclosed wherein A2 is hydrogen; A1 is 
L1 and L5 are S(O)2; L2 is N(R11); L4 is a covalent bond; L6 is N(R15); R1, R3, R4, R5, R6, R8, R9, and R10 are hydrogen; R2 and R7 are independently selected from hydroxy, alkoxy, or xe2x80x94NZ1Z2; L3 is C(xe2x95x90CHC(O)OR12); and Z1, Z2, R11, R12, and R15 are as defined in formula (I).
In another embodiment of the present invention, compounds of formula (I) are disclosed wherein A2 is hydrogen; A1 is 
L1 is N(R11); L5 is N(R15); L2 and L6 are xe2x80x94S(O)2; L4 is a covalent bond; and L3, R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R11, and R15 are as defined in formula (I).
In another embodiment of the present invention, compounds of formula (I) are disclosed wherein A2 is hydrogen; A1 is 
L1 is N(R11); L5 is N(R15); L2 and L6 are xe2x80x94S(O)2; L4 is a covalent bond; R1, R3, R4, R5, R6, R8, R9, and R10 are hydrogen; R2 and R7 are independently selected from hydroxy, alkoxy, or xe2x80x94NZ1Z2; L3 is C(O); and Z1, Z2, R11, and R15 are as defined in formula (I).
In another embodiment of the present invention, compounds of formula (I) are disclosed wherein A2 is hydrogen; A1 is 
L1 and L5 are S(O)2; L2 is N(R11); L6 is N(R15); L3 and L4 are independently selected from C(O) or C(xe2x95x90NOR13); R13 is hydrogen; and R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R11, and R15 are as defined in formula (I).
In another embodiment of the present invention, compounds of formula (I) are disclosed wherein A2 is hydrogen; A1 is 
L1 and L5 are S(O)2; L2 is N(R11); L6 is N(R15); L3 and L4 are independently selected from C(O) or C(xe2x95x90NOR13); R13 is hydrogen; R1, R3, R4, R5, R6, R8, R9, and R10 are hydrogen; R2 and R7 are independently selected from hydroxy, alkoxy, or xe2x80x94NZ1Z2; and Z1, Z2, R11, and R15 are as defined in formula (I).
In another embodiment of the present invention, compounds of formula (I) are disclosed wherein A1 is hydrogen; A2 is 
L1 and L5 are S(O)2; L2 is N(R11); L6 is N(R15); L3 and L4 are independently selected from C(O) or C(xe2x95x90NOR13); R13 is hydrogen; and R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R11, and R15 are as defined in formula (I).
In another embodiment of the present invention, compounds of formula (I) are disclosed wherein A1 is hydrogen; A2 is 
L1 and L5 are S(O)2; L2 is N(R11); L6 is N(R15); L3 and L4 are independently selected from C(O) or C(xe2x95x90NOR13); R13 is hydrogen; R1, R3, R4, R5, R6, R8, R9, and R10 are hydrogen; R2 and R7 are independently selected from hydroxy, alkoxy, or xe2x80x94NZ1Z2; and Z1, Z2, R11, and R15 are as defined in formula (I).
Another embodiment of the present invention relates to pharmaceutical compositions comprising a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof in combination with a pharmaceutically acceptable carrier.
Another embodiment of the present invention relates to a method for treating pain in a mammal in need of such treatment comprising administering to the mammal a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof in combination with a pharmaceutically acceptable carrier.
Another embodiment of the present invention relates to a method of treating urinary incontinence in a mammal in need of such treatment comprising administering a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof.
Another embodiment of the present invention relates to a method of treating bladder overactivity in a mammal in need of such treatment comprising administering a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof.
As used throughout this specification and the appended claims, the following terms have the following meanings:
The term xe2x80x9calkenylxe2x80x9d as used herein, means a straight or branched chain hydrocarbon containing from 2 to 10 carbons and containing at least one carbon-carbon double bond formed by the removal of two hydrogens. Representative examples of alkenyl include, but are not limited to, ethenyl, 2-propenyl, 2-methyl-2-propenyl, 3-butenyl, 4-pentenyl, 5-hexenyl, 2-heptenyl, 2-methyl-1-heptenyl, and 3-decenyl.
The term xe2x80x9calkoxyxe2x80x9d as used herein, means an alkyl group, as defined herein, appended to the parent molecular moiety through an oxygen atom. Representative examples of alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy, tert-butoxy, pentyloxy, and hexyloxy.
The term xe2x80x9calkoxycarbonylxe2x80x9d as used herein, means an alkoxy group, as defined herein, appended to the parent molecular moiety through a carbonyl group, as defined herein. Representative examples of alkoxycarbonyl include, but are not limited to, methoxycarbonyl, ethoxycarbonyl, and tert-butoxycarbonyl.
The term xe2x80x9calkoxycarbonylalkylxe2x80x9d as used herein, means an alkoxycarbonyl group, as defined herein, appended to the parent molecular moiety through an alkyl group, as defined herein. Representative examples of alkoxycarbonylalkyl include, but are not limited to, 3-methoxycarbonylpropyl, 4-ethoxycarbonylbutyl, and 2-tert-butoxycarbonylethyl.
The term xe2x80x9calkylxe2x80x9d as used herein, means a straight or branched chain hydrocarbon containing from 1 to 10 carbon atoms. Representative examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, and n-decyl.
The term xe2x80x9calkylcarbonylxe2x80x9d as used herein, means an alkyl group, as defined herein, appended to the parent molecular moiety through a carbonyl group, as defined herein. Representative examples of alkylcarbonyl include, but are not limited to, acetyl, 1-oxopropyl, 2,2-dimethyl-1-oxopropyl, 1-oxobutyl, and 1-oxopentyl.
The term xe2x80x9calkylcarbonyloxyxe2x80x9d as used herein, means an alkylcarbonyl group, as defined herein, appended to the parent molecular moiety through an oxygen atom. Representative examples of alkylcarbonyloxy include, but are not limited to, acetyloxy, ethylcarbonyloxy, and tert-butylcarbonyloxy.
The term xe2x80x9ccarbonylxe2x80x9d as used herein, means a xe2x80x94C(O)xe2x80x94 group.
The term xe2x80x9ccarboxyxe2x80x9d as used herein, means a xe2x80x94CO2H group.
The term xe2x80x9ccarboxyalkylxe2x80x9d as used herein, means a carboxy group, as defined herein, appended to the parent molecular moiety through an alkyl group, as defined herein. Representative examples of carboxyalkyl include, but are not limited to, carboxymethyl, 2-carboxyethyl, and 3-carboxypropyl.
The term xe2x80x9chaloxe2x80x9d or xe2x80x9chalogenxe2x80x9d as used herein, means xe2x80x94Cl, xe2x80x94Br, xe2x80x94I or xe2x80x94F.
The term xe2x80x9chaloalkoxyxe2x80x9d as used herein, means at least one halogen, as defined herein, appended to the parent molecular moiety through an alkoxy group, as defined herein. Representative examples of haloalkoxy include, but are not limited to, chloromethoxy, 2-fluoroethoxy, trifluoromethoxy, 2-chloro-3-fluoropentyloxy, and pentafluoroethoxy.
The term xe2x80x9chaloalkylxe2x80x9d as used herein, means at least one halogen, as defined herein, appended to the parent molecular moiety through an alkyl group, as defined herein. Representative examples of haloalkyl include, but are not limited to, chloromethyl, 2-fluoroethyl, trifluoromethyl, pentafluoroethyl, and 2-chloro-3-fluoropentyl.
The term xe2x80x9chydroxyxe2x80x9d as used herein, means an xe2x80x94OH group.
Compounds of the present invention were named by ACD/ChemSketch version 5.0 (developed by Advanced Chemistry Development, Inc., Toronto, ON, Canada) or were given names which appeared to be consistent with ACD nomenclature.
Representative compounds of the present invention include, but are not limited to:
9-(hydroxyimino)-N,Nxe2x80x2-bis(3-hydroxyphenyl)-9H-fluorene-2,7-disulfonamide;
{[(2,7-bis{[(3-hydroxyphenyl)amino]sulfonyl}-9H-fluoren-9-ylidene)amino]oxy}acetic acid;
9-hydroxy-N,Nxe2x80x2-bis(3-hydroxyphenyl)-9H-fluorene-2,7-disulfonamide;
9-hydrazono-N,Nxe2x80x2-bis(3-hydroxyphenyl)-9H-fluorene-2,7-disulfonamide;
(2,7-bis{[(3-hydroxyphenyl)amino]sulfonyl}-9H-fluoren-9-ylidene)acetic acid;
N-(3-{[(9-(hydroxyimino)-7-{[(3-hydroxyphenyl)amino]sulfonyl}-9H-fluoren-2-yl)sulfonyl]amino}phenyl)acetamide;
N-(3-aminophenyl)-9-(hydroxyimino)-Nxe2x80x2-(3-hydroxyphenyl)-9H-fluorene-2,7-disulfonamide;
9-(hydroxyimino)-N,Nxe2x80x2-bis(3-hydroxyphenyl)-N-methyl-9H-fluorene-2,7-disulfonamide;
N,Nxe2x80x2-bis(3-hydroxyphenyl)-9-(methylhydrazono)-9H-fluorene-2,7-disulfonamide;
N,Nxe2x80x2-bis(3-hydroxyphenyl)-9,10-dioxo-9,10-dihydro-2,7-anthracenedisulfonamide;
10-(hydroxyimino)-N,Nxe2x80x2-bis(3-hydroxyphenyl)-9-oxo-9,10-dihydro-2,7-anthracenedisulfonamide;
9,10-bis(hydroxyimino)-N,Nxe2x80x2-bis(3-hydroxyphenyl)-9,10-dihydro-2,7-anthracenedisulfonamide;
N,Nxe2x80x2-bis(3-hydroxyphenyl)-9,10-dioxo-9,10-dihydro-2,6-anthracenedisulfonamide;
10-(hydroxyimino)-N,Nxe2x80x2-bis(3-hydroxyphenyl)-9-oxo-9,10-dihydro-2,6-anthracenedisulfonamide;
9,10-bis(hydroxyimino)-N,Nxe2x80x2-bis(3-hydroxyphenyl)-9,10-dihydro-2,6-anthracenedisulfonamide;
3-methoxy-N-(7-{[(3-methoxyphenyl)sulfonyl]amino}-9-oxo-9H-fluoren-2-yl)benzenesulfonamide;
3-hydroxy-N-(7-{[(3-hydroxyphenyl)sulfonyl]amino}-9-oxo-9H-fluoren-2-yl)benzenesulfonamide; and
3-hydroxy-N-(9-(hydroxyimino)-7-{[(3-hydroxyphenyl)sulfonyl]amino}-9H-fluoren-2-yl)benzenesulfonamide; or pharmaceutically acceptable salts, amides, esters, or prodrugs thereof.
Abbreviations which have been used in the descriptions of the Schemes and the Examples that follow are: DMSO for dimethylsulfoxide; HPLC high pressure liquid chromatography; and THF for tetrahydrofuran.
The compounds and processes of the present invention will be better understood in connection with the following synthetic Schemes and Examples which illustrate a means by which the compounds of the present invention can be prepared. 
Fluorenes of general formula (3), wherein R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R11, and R15 are as defined in formula (I), can be prepared as described in Scheme 1. 9-Oxo-9H-fluorene-2,7-disulfonyl dichloride, purchased from Maybridge, can be treated with anilines of general formula (I) and (2) to provide fluorenes of general formula (3). 
Fluorenes of general formula (5), (7), and (9), wherein R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R11, R12, R13, R14, and R15 are as defined in formula (I), can be prepared as described in Scheme 2. Fluorenes of general formula (3) can be treated with amines of general formula (4) and a catalytic amount of acid in an alcoholic solvent such as, but not limited to, ethanol with heat to provide fluorenes of general formula (5).
Fluorenes of general formula (3) can be treated with hydrazines of general formula (6) and a catalytic amount of acid in an alcoholic solvent such as, but not limited to, ethanol with heat to provide fluorenes of general formula (7).
Fluorenes of general formula (3) can be treated with phosphonium reagents of general formula (8) under standard Wittig conditions to provide fluorenes of general formula (9). Fluorenes of general formula (3) can also be treated with phosphonates under standard Homer-Wadsworth-Emmons conditions to provide fluorenes of general formula (9). 
Fluorenes of general formula (14), wherein R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R11, and R15 are as defined in formula (I), can be prepared as described in Scheme 3. Fluorenes of general formula (11), prepared as described in P. J. Perry, M. A. Read, R. T. Davies, S. M. Gowan, A. P. Reszka, A. A. Wood, L. R. Kelland, S. Neidle, J. Med. Chem. 1999, 42, 2679, can be treated with sulfonyl chlorides of general formula (12) and (13) in a solvent or cosolvent such as, but not limited to, THF and pyridine to provide fluorenes of general formula (14). 
Anthracenes of general formula (16), wherein R1, R2, R3, R4, R5, R6, R7, R8, R9, and R10 are as defined in formula (I), can be prepared as described in Scheme 4. Anthraquinone-2,7-disulfonic acid disodium salt, can be treated with phosphorous oxychloride to provide anthracenedisulfonyl dichloride. Anthracenedisulfonyl dichloride can be treated with anilines of general formula (1) and (2) to provide anthracenes of general formula (16).
Anthraquinone-2,6-disulfonic acid disodium salt can be treated as described in Scheme 4 to provide 2,6-disubstituted anthracenes.
Anthracenes of general formula (16) can be treated as described in Scheme 2 to provide oximes, hydrazones, xcex1,xcex2 unsaturated esters, or xcex1,xcex2 unsaturated acids.
The following Examples are intended as an illustration of and not a limitation upon the scope of the invention as defined in the appended claims.